Molecular Mechanisms Of GOLPH3-PI4P Signaling Pathway Regulating Autophagy

Autophagy delivers damaged,aged,or unnessary macromolecules or organelles to lysosomes for degradation,while releasing free small molecules for cellular recycling,thereby maintaining cellular homeostasis.The process of autophagy responds to internal and external cues and is regulated by multiple cellular signaling pathways.Typically,autophagy includes initiation,growth,maturation,and fusion of autophagosomes with lysosomes.Therefore,autophagy is a finely regulated process involving multiple molecules.GOLPH3 is an oncogene that is overexpressed in various solid tumors.It localizes to the Golgi apparatus through binding to PI4 P enriched there,and its oncogenic activity is associated with activation of the m TOR signaling pathway.PI4 P is distributed in the cell membrane,Golgi apparatus,lysosomes,and endosomes,and exerts important physiological effects by binding to its effector proteins.Since the m TORC1 signaling pathway plays an important role in the regulation of autophagy,here,we mainly focuses on the molecular mechanism of GOLPH3 in regulating autophagy.Our results showed that GOLPH3 knockdown led to an increase in the levels of LC3-II/LC3-I and an increase in the number of double-membrane autophagosomes.At the same time,the level of autophagic substrate p62 also increased,indicating that knockdown of GOLPH3 promotes autophagy initiation but impairs autophagic degradation.Autophagy flux analysis also showed that knockdown of GOLPH3 affected the fusion of autophagosomes with lysosomes or altered the acidic environment of normal lysosomes.Knockdown of GOLPH3 reduced m TORC1 activity,and lowering intracellular PI4 P levels by knocking down PI4K2α and PI4K3β could rescue the decrease in m TORC1 activity caused by GOLPH3 knockdown.In addition,knockdown of GOLPH3 could cause the transcription factor TFEB to remain in the nucleus,promoting lysosomal biogenesis.Knockdown of GOLPH3 impaired the activity of the lysosomal acid hydrolases CTSD and CTSB,resulting in a decrease in the number of acidic lysosomes and decreased lysosomal autophagic degradation.When PI4K2α and PI4K3β,which are located on the Golgi apparatus,were knocked down to decrease PI4 P levels in GOLPH3 knockdown cells,it was found that the defects such as an increase in the overall number of lysosomes,a decrease in the number of acidic lysosomes,and a decrease in the activity of hydrolases caused by GOLPH3 knockdown could be restored.In summary,the results of this study demonstrate that GOLPH3 can regulate the m TORC1 signaling pathway and nuclear-cytoplasmic localization of the transcription factor TFEB,which has an impact on lysosomal biogenesis.We further confirmed that GOLPH3 can also regulate the number of acidic lysosomes and the activity of the hydrolases CTSD and CTSB,thereby affecting autophagic degradation.Moreover,the effects of GOLPH3 on the m TORC1 signaling pathway and lysosomal activity both depend on the level of the PI4 P.Therefore,our work reveals the molecular mechanism by which the GOLPH3-PI4 P signaling pathway regulates cellular autophagy,providing a theoretical basis for further understanding the role of GOLPH3 in the development of tumors and the screening of anti-tumor drugs targets.